(79b) Accelerating the Design and Engineering of Microbial Cell Factories Via Synthetic Biology and Automation

Pu Xue,¹ Tong Si,^1,2 Stephan Lane,²Huimin Zhao^1,2,★

¹Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801; ²Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801

^★Correspondence should be addressed to H.Z. (zhao5@illinois.edu). Phone: (217) 333-2631. Fax: (217) 333-5052.

http://scs.illinois.edu/~zhaogrp/

Abstract

Microbial cell factories (MCFs) have been increasingly engineered for production of chemicals and fuels. However, metabolic engineering of MCFs is still time-consuming and labor-intensive. To address this limitation, we attempt to perform genetic engineering and phenotypic screening by integrating the Design-Build-Test-Learn (DBTL) cycle with a biofoundry. As proof-of-concept, we developed a mass spectrometry based high-throughput screening method for detecting medium-chain fatty acids (MCFAs) in Saccharomyces cerevisiae by using lipids as a proxy. Four 1^st shell key residues in the fatty acid synthase were chosen based on computational modeling. Site-saturation mutagenesis was performed at each residue to create 4 libraries of interest and 288 colonies were screened in total to achieve 95% library coverage. The results from mass spectrometry indicated increased shorter acyl-chain phosphatidylcholine (PC) production in ~100% mutants compared with wild type. We additionally attempted to develop automated multiplexed gene knock-outs or overexpression and genome-scale engineering of S. cerevisiae using our biofoundry. We first assembled individual modules together into an integrated automated pipeline. Next, as a demonstration, we created 10 transcriptional factor knock-outs in S. cerevisiae for improving acetic acid tolerance. Through our newly developed multimodal, multiscale acoustic robotic mass spectrometry, the throughput of screening can achieve up to 2 colonies/liquid cultures per second. These technologies should be generally applicable for metabolic engineering of MCFs for production of fatty acids, fatty alcohols, biofuels and chemicals.